This invention relates generally to improving the performance and reliability of test strips employing moisture-sensitive reagents, particularly those used to determine the presence of analytes in urine samples, such as albumin, protein, creatinine, nitrate, uristatin, leukocyte esterase (white blood cells), occult blood (red blood cells), ketones, glucose, bilirubin, urobilogen and others familiar to those skilled in the art. Measurement of proteases and protease inhibitors such as leukocyte esterase (human elastase) or urinary trypsin inhibitor (Bikunin, Uristatin) are especially important in that they may indicate infections of the kidney or urogenital tract. These proteases and protease inhibitor strips rely on hydrolysis of proteolytic substrates by proteases to generate detectable signals. Since the biochemical reactions require water for enzymatic hydrolysis to occur, they tend to be sensitive to moisture and may interfere with the detectable signals through background reactions.
U.S. Pat. No. 5,663,044 is incorporated herein by reference. This patent describes the background art in the field of detecting leukocyte, esterase or protease and for teaching generally the use of a composition including a diazonium salt, one of a group of esters subject to hydrolysis in the presence of leukocyte, esterase or protease and an alkaline earth metal. The '044 patent teaches the measuring of reflectance of light at about 570 nm to determine the amount of leukocytes in the urine sample. The reflectance at 570 nm is compared with a standard measurement of reflectance at 690 nm. The patent indicates that the presence of alkaline earth metal promotes stability of the diazonium salt. It also suggests that the alkaline earth metal absorbs moisture which can cause background color change. Experience has shown that this reagent system is sensitive to moisture and that the test strips must be kept in a dry environment before use. The moisture content of test strips should be kept below 2 wt % to avoid degrading performance. However, measuring moisture in test strips is not very accurate. There is a particular need to measure moisture content very accurately to assure that test strips are stable in a closed dry container over several years.
Another example of the use of moisture-sensitive reagents is discussed in U.S. Pat. Nos. 6,770,764; 6,955,921; and 7,001,737 incorporated herein by reference. In the reactions described, the presence of urinary trypsin inhibitors in a urine sample is detected by adding a sample to test strips containing a known amount of trypsin, a trypsin substrate, i.e. arginine esters hydrolyzed by trypsin to produce an alcohol, and a diazonium salt. When trypsin inhibitors are present they inhibit the reaction between trypsin and the substrate. This reduces the color produced by diazonium salt from its reaction with the phenol produced when the trypsin substrate reacts with the known amount of trypsin. By measuring the color produced, the presence of trypsin inhibitors can be determined.
In U.S. Pat. No. 6,316,264 an infra-red (IR) dye is added to a predetermined location on reagent strips in order to assure that the strips are properly aligned in the instrument used to detect and/or measure the presence of analytes in a sample applied to the strip. The IR range for the dyes was broadly between 700 and 2500 nm, but dyes having strong absorbance in the range of 825-855 nm were said to be preferred, with absorbance in the visible range (400-700 nm) less than 20%.
As suggested above, it would be advantageous if urine test strips that are moisture-compromised could be identified with improved accuracy to prevent unsafe use and to correct for moisture-affected results. Since reagents which involve hydrolysis of proteolytic substrates are especially sensitive to moisture, if the reagents themselves could be used to indicate the presence of undesirable moisture, significant improvement would have been obtained. The present invention provides such a method, which will be described in detail below.